Thursday, April 14
1:30 p.m-3:00 p.m.
Session 7:
Pollution Source Identification,
Tracking, and Sanitary Surveys
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f*"!
Day Two: Session 7
J
iL2a
Florida Beach Geomorphology and
Beach Management Practices Correlate
with Enterococci Percent Exceedances
Helena Solo-Gabriele
University of Miami
Abstract
Fecal indicator bacteria (FIB) data for
Florida's 316 beaches that are monitored through
the Florida Healthy Beaches Program were eval-
uated to identify possible correlations between
FIB levels and beach physical characteristics
and beach management practices. Beach char-
acteristics were evaluated for coastal hydrody-
namics using offshore bouy information and
for observable features using Google imagery
Management information was obtained through
a survey that was sent to beach managers.
Results show that beach geomorphology (open
coast versus bay and beach width), wave energy
proximity of rivers/canals/marshes, presence
of piers/causeways, and degree of surrounding
urbanization was correlated with FIB levels.
For open coast beaches there was a statistically
higher level of enterococci percent exceedence
for beaches that display higher levels of urban-
ization (p=0.03), contain major human con-
structions like piers and/or causeways (p=0.03),
and were narrower in width. Beaches within
bays or reside in close proximity to marshes
were also characterized by higher enterococci
percent exceedences (p<0.01). From the beach
management survey we also found that beaches
that charged fees had lower FIB levels (p<0.01).
Beaches that allowed dogs had higher FIB levels
(although borderline not statistically significant,
p=0.07). Results suggest that the cumulative
effects of beach management practices can play
a role in whether beaches comply with EPA
recreational water quality guidelines.
Biosketch
Dr. Helena Solo-Gabriele is a professor
in the Department of Civil, Architectural, and
Environmental Engineering at the University of
Miami's College of Engineering as well as the
college's associate dean for research. Dr. Solo-
Gabriele's research has focused on evaluating
the relationship between the environment
and human health and has spanned diverse
areas that include evaluating the human health
impacts of metals/pesticides in the environment
and of microbial contaminants in coastal zones.
Her microbial contaminants research focuses
on evaluating the water-sand interface and the
microbial contaminants in that area as well
as the conditions under which microbes are
released from that zone. Most recently, Dr. Solo-
Gabriele has focused her efforts on evaluating
data for many beaches; her presentation will
summarize her work on beaches throughout the
State of Florida.
121
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U.S. EPA's 2016 Recreational Waters Conference
Florida Beach Geomorphology and Beach
Management Practices Correlate with
Enterococci Percent Exceedences
OHH
Elizabeth Allison Ryan Sabina bathvika . . ,
•telly Donahue Munlatalu RoUhimbckovo Ramaji ACKP OWlSU^rYl ©HlS
Outline/Methods
• Florida's Beach Monitoring Program
• Florida's Beach Geomorphology
- Wind Data and Beach Slopes (Feng et al.)
- Visual from Google Earth (In progress)
Association
• Beach Management
""with
- .Survey. Sewage, stormwater, solid waste,
Enterococci
human and animal densities, seaweed
and grooming, fees (In progress)
• Conclusions & Recommendations
Florida Healthy
Beaches Data
• Period of Record: 2000 to 2014 (2015)
• Sampling Frequency: Monthly Weekly Variabfe
• Converted FIB measures to "percent exceedences"
FIB
n
Threshold
for % exceed
Enterococci
182,000
104 CFU/100 ml
Fecal Coliform 154,000 400 CFU/100 ml
• No. Points per Beach: 120 to 690 (enterococci)
• No. Beaches: 316
Spatial Trends
! Nassau
M"™a -J jjSl
Big Bend . •
bL -MM
1 * , ,
Percent Exceedences
• """
j •
Tempo Doy • ,
E • 1
V * 1
1
^ >2to6
¦ Miami J
1V J
enterococci
^ s-v fl Keys
Spatial Trends
122
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Marsh Beaches'
Bay Beach
Carabelle Beach
Open
Coast
Beaches
George Island, 11"> Sttc.
St. Georee Island. Franklin Street
Nassau
Cscambla
Other Factors
(In process)
Factor
1 % exceedence 1
P
\wm
Pier
6.3
2.8
<0.001
(n-64)
(n—251)
Pier
2.2
l.b
U.1U2
(Open coast only)
(23)
(186)
Causeway
5.6
3.8
0.022
(23)
(293)
Causeway
6.2
6.5
0.830
(Bay only)
(16)
(52)
Rivers w/ln 2000'
9.0
2.3
0.010
(Excludes bays)
(24)
(226)
Canals w/in ?00(Y
81
3 R
0 017
(11)
(3Ub)
fnrrplatinn witf
% exceedence
Beach Width
(Open coast only)
UlbdllUdliUII
(Open coast only)
St. George Island — Franklin Blvd
Dog Densities (% exceed)
Q
P-value
All Types
4.1
(n=55)
2.8
(144)
0.02
Open Coast 2.3 1.5 e'O.Ol
(25) (104)
Seaweed condition (%exceed)
Beach Type
Dense/
Medium
Zero P-value
All Types
7A
3.2 0.02
m
(?q)
Open Coast
3.8
0.4 <0.01
(20)
(24)
Other Factors - Beach Management
(In process)
Beach Management Practices (Survey-97%)
123
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U.S. EPA's 2016 Recreational Waters Conference
Conclusions:
(Factors Associated With Low Bacteria)
Hydrodynamics (Feng et al. 2016)
• High Wave Energy, Steeper Slopes
Physical Features
• Open Coast_< Ba^< Marsh
• No Piers, No Causeways, Low Urban
• Wide Beaches
• Rivers/Canals Far Away
Beach Management
• No Dogs
• No Seaweed
• Charge. Fees
• Covered TrashcanS -
Recommendations
1) Evaluate high enterococci relative to fecal coliform
2) Evaluate sand quality.
3) Evaluate impact of water chemistry at marsh beaches.
124
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f*"!
Day Two: Session 7
J
iL2a
Source Tracking of Ankle-Depth Water
Quality Impairment at Freshwater
Beaches in Ontario, Canada
Thomas Edge, PhD
Environment Canada
Abstract
Ankle-depth E. coli concentrations were
investigated at 15 freshwater beaches in south-
ern Ontario, Canada. Across about 1,300 sam-
pling events, ankle-depth E. coli concentrations
were found to exceed chest-depth E. coli concen-
trations most often at sheltered beaches (91%),
followed by exposed beaches (84%) and river
beaches (68%). Ankle-depth E. coli concentra-
tions could exceed 10,000 colony-forming units
(CFUs) / 100 milliliters (mL) at times at 10 of
the 15 beaches, and two sheltered beaches had
ankle-depth E. coli concentrations that could
exceed 100,000 CFU / 100 mL. Pair-wise analy-
ses found that ankle-depth E. coli concentrations
were significantly higher than adjacent chest-
depth concentrations at each of the 15 beaches
investigated (p < 0.05). Microbial and chemical
source tracking techniques are being applied
to assess the potential significance of this water
quality impairment. Application of a suite of
microbial assays (e.g., endpoint and qPCR fecal
marker assays for human, gull) and chemi-
cal assays (e.g., caffeine, carbamazepine) have
indicated that bird fecal contamination rather
than human fecal contamination is the primary
fecal pollution source at a number of beaches.
Additional research is required to better under-
stand the potential for exposure in ankle-depth
waters at freshwater beaches and the potential
for health risks.
Biosketch
Dr. Tom Edge is a research scientist at
Environment and Climate Change Canada's
National Water Research Institute in Burlington,
Ontario. He received his doctorate in environ-
mental microbiology from Carleton University
in Ottawa, his master of science degree from the
University of Ottawa, and his bachelor science
degree in marine biology from the University of
Guelph. Dr. Edge conducts research on micro-
bial source tracking and waterborne pathogens
in source waters used for drinking, recreation,
and shellfish production across Canada. He
has served as president and a board member
of the Great Lakes Beach Association and has
been a scientific advisor to the International
Joint Commission and Health Canada on beach
issues and Canadian recreational water quality
guidelines.
125
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U.S. EPA's 2016 Recreational Waters Conference
Sources of Enterococci and their
Relation with Environmental Factors at
an Inland Beach
Asli Asian, PhD
Georgia Southern University
Abstract
Nonpoint sources of pollution have been
reported as an alternative source of high entero-
cocci levels in beaches. In this study, persistent
high levels of enterococci were investigated at an
inland beach in Georgia that has been under per-
manent advisory since 2005. On a monthly basis
for 1 year, data were collected on Enterococci
(using method 1600 for water, Enterolert for
sediment, and method 1611 for water and sedi-
ment), source tracking markers (HF183 and GFD),
temperature, salinity, pH, dissolved oxygen, and
turbidity. Microbial source tracking analyses
showed high HF183 concentrations around the
beach at the beginning of the study (405 cali-
brator cell equivalents/100 milliliters [ml]) and
further investigations proved an illicit sewage
leak close to the beach. Culturable enterococci in
water significantly decreased below 70 CFU/100
ml criteria value and HF183 was below detec-
tion limits after controlling the sewage intrusion
to the beach. In addition, wildlife (particularly
avian species) was a major source of enterococci
in this area. GFD marker ranged from 152-1077
gene copies/100 ml in HF183 negative water sam-
ples. Sediment also harbored culturable entero-
cocci and concentrations ranged from 29-24196
MPN/100 ml throughout the study, showing
significant relation to temperature (p < 0.001).
Tidal movements and precipitation were the
major factors (p < 0.001) for elevated concentra-
tions of enterococci after controlling the point
source. Further studies are needed to investigate
the relation between culturable enterococci and
pathogens such as enteric viruses to assess health
risks upon exposure to contaminated sediments
on inland beaches.
Biosketch
Dr. Asli Asian is a water microbiologist
whose primary research areas include micro-
bial source tracking, health risk assessment of
water resources, and ecology of pathogens in
the aquatic environment. Her research program
bridges ecosystems and public health. Dr. Asian
is currently an assistant professor at the
Department of Environmental Health Sciences
at Georgia Southern University. She holds
doctoral and master of science degrees from
Istanbul University, Institute of Marine Sciences
and Management, and has completed her post-
doctoral studies at Michigan State University,
College of Natural Resources and Agriculture.
Dr. Asian has conducted and participated
in several health-related water microbiology
projects in Turkey, Malawi, Ghana, the United
States, and other countries; authored various
manuscripts; and presented at numerous con-
ferences. She has been serving in various state
and federal agencies and organizations as an
advisor, reviewer, scientific committee member,
and/or affiliated faculty member.
126
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13
Day Two: Session 7
Sources of enterococci and
their relation with
environmental factors at an
inland beach
ASLI ASLAN
GEORGIA SOUTHERN UNIVERSITY
aaslan@seorHiasouthern.edu
Disclaimer
This presentation was prepared by Dr. Asian under grant award
#NA14NOS41Qft117tothp Hppartmpnt of Natural rpsnnrrpsfrom thp Offirp nf
Ocean and Coastal Management, National Oceanic and Atmospheric
Administration. The statements, findings, conclusion and recommendations arc
those the author(sJ and do not necessarily reflect the views of DNR, OCRM or
NOAA.
Krffc ass
Coastal Georgia
•3,400 miles of tidal shoreline
•100 miles of sand hearh
•300 square miles of open Atlantic Ocean
*9 major esluaries
^ J|
•14 barrier islands
9
l':- ' 3
FniirtPiiy nf Ms Flinfvth Chpripy
Kings Ferry Beach
Under permanent advisory since 2005.
127
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U.S. EPA's 2016 Recreational Waters Conference
Sample Collection
Water samples: 14 sites monthly in winter,
twice a day (low-high tide) during the
summer
Sediment samples: 6 sites monthly, 14 sites
onp in winter, one in thp summer
Hearh sanitary surveys
Temperature, salinity, dissolved oxygen, pH,
turbidity
L
Enterococci
(MPN/100 ml)
Enterococci 23S
(CCE/100 ml)
HF183
(i.ufjie>/100 ml)
Kb 18
4.b43
22.23b
l.bbb.3/3
KF7fl
Q1
qsn
3m
KF3
49
1,350
142
Culture versus qPCR
Before
R* .0.76533
~ /
I 5000
"vm
After
128
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Day Two: Session 7
I I.I
KF4 KFS
¦Ent2JS ¦MF141 BGFD
Environmental Factors-Tides
SuhnrHinflt.fi Predictions
. L-JQjj i
r X / - \
1 \ :/ \
\ \ \ / :\...
\ j \ / \ / \
; ^
83/13 03/14 83/14
6|» 19m 6m
Date/Tine (LST/LDT)
83/14 83/14 83/15
12pn 6p« IQan
https^/ti desandmrrents.noaa.gov
Enterococci and Tides
i > i i
mi
Enterococci species
A total of 55 Enterococci isolates from mEI agar were
analyzed by Sanger sequencing.
Enterococci spp.
faccala
® C. faechjrn
l£ eassttiffavui
Environmental Factors-
Temperature
Sediment
Enterococci
yl—
-—
1i ..ill
ill
OCT NOV DEC IAN FEB MAR APR MAY JUN JUL AUG SEP
Months
Conclusions
•Method 1G00 and Method 1G11 results were significantly related in the
presence ot fresh sewage (p<(J.001).
•Microbial source tracking (HF183 and GFD) ib a powerful tool lo identify the
sources of high levels of Enterococci in rural environments.
• Inform Inral agpnrip«; fnr thp advantagpnn<; nf dpw tprhnnlngy
•Environmental factors such as temperature and tidal movements are drivers
for high levels of Enterococci.
• Each beach is unique, ebb or flow tide can transport Enterococci depending on
the location of the potential bourceb.
•Sediment harbors Enterococci and potentially acts as a reservoir in warmer
temperatures.
•Future studies will Involve detecting changes in the bacterial community
structure and Enterococci diversity before and after the sewage leak.
129
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U.S. EPA's 2016 Recreational Waters Conference
Acknowledgements
A
c
OUUAI RROHRCK mVKION
Elizabeth Cheney
Jill Andrews
Stefanie Nagid
Emily Markesteyn
William Pitts
Clark Alexander
Asian Lab @GeorgiaSouthern: jMp
Asheley Poole . i
Kendall Anderson
Spencer Barron
Jessica Sexton
cirouc.iA I
A
SkkU liulllulc
tXi-jnujyapliy
;w
HP
OGEECHEE 11 L " ]
KivtKKttptR I 'J m m
130
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f*"!
Day Two: Session 7
J
iL2a
Temporal Stability of Fecal Bacteria and
Microbial Source Tracking Markers in Water:
Implications for Source Allocation Using MST
Markers
Mia Mattioli, PhD
Emory University
Abstract
A coupled field and laboratory study of the
temporal stability of fecal bacteria was assessed
to inform the use of microbial source tracking
(MST) markers for inferring sources of fecal
contamination in natural waters. The temporal
stability of enterococci, E. coli, and human-
associated markers of fecal pollution was
assessed in seawater. Seawater from Half Moon
Bay California, was seeded with raw sewage
and placed into 6-8-kilodalton dialysis bags.
Approximately 30 dialysis bags were deployed
at both the surface of the water column and at
1-meter depth and destructively sampled over
10 days during the summer and winter. A dis-
tinct difference in decay at surface and at depth
was observed, presumably due to decreasing
UVA and UVB wavelength intensity at depth.
The absorbance of the ambient water illustrates
that the water column is not colored and rela-
tively clear. A model was developed for the
depth-dependent inactivation rate of the fecal
indicator bacteria (FIB) and human markers,
and it shows how a depth averaged inactivation
rate varies with depth of the water column. The
decay of MST markers and FIB was the same
in dark water and diverged in shallow waters.
This finding suggests that the ratio method for
source allocation using MST markers can be
applied only in dark water.
Biosketch
Dr. Mia Catharine Mattioli is a joint post-
doctoral scholar at Emory University and the
Waterborne Disease Prevention Branch of the
Centers for Disease Control and Prevention
(CDC). She has a bachelor of science degree
in biological engineering from the University
of Georgia (UGA) and masters and doctoral
degrees in environmental engineering from
Stanford University. Dr. Mattioli's research is
focused on the intersection between the envi-
ronment and human health with a specific inter-
est in the relationship between fecal indicators
and enteric pathogens in various environmental
matrices. During a postdoctoral fellowship at
Stanford University, she examined the decay
characteristics of fecal indicators and sewage-
related bacterial communities in environmental
waters. Dr. Mattioli is currently working on a
collaborative project between the CDC, UGA,
and Emory studying the use of ultrafiltration as
a means for monitoring microbial contamina-
tion of irrigation waters.
131
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U.S. EPA's 2016 Recreational Waters Conference
Exploring the Microbiome for New
Approaches to Microbial Source
Tracking
Sandra McLellan
University of Wisconsin—Milwaukee
Abstract
Fecal microbiome studies offer new oppor-
tunities to identify alternative indicators for
microbial source tracking that are host-associ-
ated and more informative than general indica-
tors that are ubiquitously present in humans
and other animals. Microbiome next generation
sequencing technologies provide hundreds of
thousands to millions of DNA sequences per
sample. While mining the data can be challeng-
ing, it is clear that host-associated patterns in
fecal microbiomes have important implications
for water quality testing. Bacteroidales and
Clostridiales are two bacterial groups that are
particularly rich in potential host-associated
indicators, including the organisms within the
family Lachnospiraceae, which has not been
explored in depth previously. We report the
development of Lachnospiraceae qPCR assays
that can be used in conjunction with established
Bacteroidales methods to quantify sewage
contamination in the Great Lakes, particularly
in respect to characterizing recreational beach
impairment. In addition to qPCR applications,
next generation sequencing strategies can
make use of distinctive host community trends
within the broad groups of Bacteroidales and
Clostridiales for the identification of fecal pol-
lution sources. As microbiome studies increase
the number of potential candidate host-asso-
ciated indicator strategies, future laboratory
validation and field studies will be critical to
identify the most applicable methods for recre-
ational water quality monitoring.
Biosketch
Dr. Sandra McLellan is a professor in the
School of Freshwater Sciences at the University
of Wisconsin-Milwaukee. Her research focuses
on studying microbial communities in the
urban environment and the implications for
human and ecosystem health. Studies include
determining the fate of pathogens in the Great
Lakes and determining the causes of beach
closings. Her laboratory employs next genera-
tion sequencing approaches to identify new
indicators of pollution and pollution impacts.
Dr. McLellan's group has identified several
promising alternative indicators within diverse
families of fecal bacteria and has developed
microbial signatures for humans, cattle, and
urban wildlife. These signatures are being
tracked in urban waters to delineate the extent
of sewage contamination and the drivers of pol-
lution. Dr. McLellan interacts with a wide range
of stakeholders, including public health officials
and water resource managers to translate basic
research into practical applications and sound
policy. She graduated from the University of
Wisconsin-Milwaukee in health sciences in 1990
and worked at Miller Brewing Company before
entering graduate school. Dr. McLellan earned
her graduate degree in environmental health at
the University of Cincinnati College of Medicine
in 1998.
132
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Day Two: Session 7
Exploring the microbiome for new
approaches for microbial source tracking
Sandra I. Mrl ellan, Ryan I Newton, len C. Fisher, and Dehra K. Dila
School of Freshwater Sciences, University of Wisconsin Milwaukee
Fecal Pollution contains a complex mixture of organisms
Fecal indicator bacteria
Rare, <5% in mammals.
Up to 50% in birds
f. coli
en terococci
Klphsiplln pnpumnriinp
Entcrobactcr cloaccac
Tecal anaerobes
95% of microbiota
Bacteroides spp.
Lachnosplraceae
Bifidobacteria
Prevotella spp
Pathogens
inLermiUenL
Salmonella
Cryptosporidium
viruses
Characterizing sewage
Sewage organisms 109 cells per ml
o
v.
•» mm .
mm
Sewage organisms 109 cells per ml
Cloning and sequencing the 16S rRNA gene- "who's there"
1000-2000 sequences is a good effort- only see the most abundant
nrganKms
• ' vS:
.O" " • • *
• • • • • • •
Sewage organisms 109 cells per ml
lllumia MiSeq or Hiseq sequencing
On average 100,000 sequences per sample
Sequencing Sewage
V6 rRNA gene
80% environmental
origin
Acinetobacter
Aewmonas
Pseudomonas
Arcobacter
Betaproteobacteria
>100,000 reads per sample
800 different taxa
VO.tHK) different sequences
20% faecal origin
Actinobactcria
Rartpmirtps
Trichococcus
Lachnospiraceae
Ruminococcaceat
Mrl ellan et al (7010) Fnvironmental Microbiology 17(9). 378-39?
V&ndeWalle et al. (2013) Environmental Microbiology 14(9). 2538-2552
133
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U.S. EPA's 2016 Recreational Waters Conference
How do we make sense of
millions of data points?
Sequence
Raw reads
Demultiplex
Merge
Quality trim
Chimeras
Identify the members
Community analysis
Assign taxonomy
Cluster into OTUs (oligotyping)
Network analysis to compare
sequences
LefSe analysis to identify biomakers
Genera of fecal anaerobes common Lo hosls
I _b i ¦ i-li
Sewage
Fisher et al App!Enviivn Micivbbl 81:7023-7033.
®§ #
SWine
£Wt J*
Sewage
ji
Deter-™
V^Gat
Chicken
Dog
^ Oliootvws shared
What is shared and what is unique?
What taxa contain preferred
and unique sequences?
Tava
Bacteroides
Dacteroides
Rn«*hi iria
Bacteroides
Bacteroides
Blautia
Aliilipti
Blautia
I arhnospirareae
Baueroldes
Bactoroidos
Blautia
Dorea
Rnwhnria
l-aecalibactenum
Bacteroides
Blautia
Baueroldes
Dorea
Spf|iipnrp
LbAbLAbAAl I ACbbCCJbIAACCLAI lb
CGGGCAACATGATGC ATGTCACCCAGT G
AfirTTrrrfiTrrrarfTrrrAfiT
CGAG CACTATGATCCG GTTCACG AGCAG
CGAGCAACATAWCAGTATACTCTGTG
AATCTCACCGTCTTTACCTCTTTGCAGT
GCGATCTAGGTCTCGG
AGTGCCACCATCCTCATCTTCTTACGCA
TGfTrCCCrAf.CGGrAAT6TCTTCCTTGAC.AGT
CGA6CAACATATGGCGCCATACGAGCGT
CGAGCAAAATTAT GCCCATACCCCATT G
AGTCrCACCACrCTCGTCFTOTACA6A
GCAACGCCTAAGCTACT
AGnrrrrfrrrTrArrrrTrAfiT
ILI ALbAIbLbALAIAI ICb
CGAGCAACATATGGCGCTATACGAGCGT
aatctcaccggactccccttcttacgga
CGA6C ATTATATAT CAGTATACTCAATG
GCAACGCCTAAGTTACT
CTGATCGAGGTCTCAG
Rankin
¦ mm
Spwagp
1
4
ft
7
¦ f Sewag.
1
9
13
16
Hshfir fit ai 'A) 1h
17
Appl Environ
Miciobiol 81:7023-
18
7033
20
21
22
Lachnosplraceae
23
very promising
74
lb
26
77
28
29
31
Compare humans, sewage, chickens and cows
fill
»kua«*DffV*J2{n.7a-!(9)
iibum mrmws (dqiwh)
Gactimnl Nmb vMKKMM (AB7I5C)
I ar.hnn?
Lachnol
McLellan et al. (2013) Environmental Microbiology
Larhnnspirareae as a qPCR target for
alternative indicators
Clone near-full length sequences
Align and design primers for qPCR
Developed assay for qPCR
Lachno2, Lachno3,and Lachnol2
Ruttrl
•' Cuuoi»>
TTACCAAATCTTGACA TCCCTCTCACCGOTCT'TAATCCGACCT'TCTCT'CGGAGCAjAGG TGACAGGTGG"
ACCCCTCT'TAXTCCCACCTTCTCT'CCCACCAjACGTGI€
;acccctct-taatcccaccttctct-cccacca;acctc*cacctcc-
:acccctct_taxtcccaccttctct'Cccagca:acgtcacacctcc"
CACCCCTCfTAATCCCACCTTCTCT-CCCACCAIACCTCACACCTCC*
CWCGClil' lAAlUGAOU I ICCI COGOAIAjAGIjAGACAGG 110
:accccacc-ta
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Day Two: Session 7
Sequence profiles of Blautia act as a fingerprint
I leatmap of sequence abundance shows humans (sewage)
and various animals have unique sequence profiles
¦ Chicken tfiOog 8 Con Human ¦ Sewage B Swine | Swine (Died) Cat Deer
Erenet al (2015) feme J 9:90-100. McLellan and Eren (2015) Trends in Microbiol 22:697-705
Sequence profiles of Blautia act as a fingerprint
Next Steps: build a searchable database so sequencing data can
hp interpreted Cost: $40-$100 for sequencing
¦ Chicken ¦ Dog |Cow Human ¦ Sewage ¦ Swine ¦ Swine (Bum) Cat Deer
Erenet al (2015) IsmeJ 9:90-100. McLellan and Eren (2015) Trends in Microbiol 22:697-706
Signature of sewage microbial communities
I Bifidobacterium
I lachnospiraceae;Roseburia
- Bactaroidas
¦ Ri imirtoror r arpap;Fapralihartp rii im
Ruminococeaeeae;Rumi no coccu*
is Lachnosoiraceae;Dorea
Junction Harbor Sao
Survey stations into Lakes Michigan
Human indicators in the nearshore
Heavy Kain/Blending tvent heavy Kain/LbU & bbU
ui b.0ti 1.5E4 1.0ES 1.0E6 2.SE7 Newlun el dl. (2013) Miuubiu! Etuluyy
Copies/L water 65:1011-23
Characterizing the gull microbiome
Gulls can be major sources of fecal contamination
Gulls have been shown to harbor high abundances of FIB,
£. coli and enterococcl, which can be detected readily as
part of routine beach monitoring
Despite the ubiquitous
presence of gull fecal
contamination at
beaches, the microbial
community is relatively
poorly characterized
135
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U.S. EPA's 2016 Recreational Waters Conference
Microbial community structure in gull fecal samples
100%
¦ Staphytocoecaceao: Staphylococcus
aCryctpelotrichacope- Timcibactor
Catellicoccus
. marimammalium
Enxerococcaceae and Enrerobacterlaceae were
identified db lite Iwu inubl abundant fdinilieb.
Microbial community structure in gull fecal samples
Knskey pt al. (2014) Appl Fm/imn Microbial 80:757-765
South Shore Beach, Milwaukee Wl
worst water quality on Lake Michigan
_ R 1
I
I
1 i
In
if "i?
¦ Fxislino beach ¦ Proposed bearh
Baseline <0-25 in rain >0.25 in
Old beach water quality 40X worse
Than prnpnsprl hparh 140 mpfprc away
Old vs. new beach alternative indicator results
Dry weather
E. coli enterococci BacHuman Lachno2 Gull2
Old vs. new beach alternative indicator results
E. coli enterococci BacHuman Lachno2 Gull2
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Day Two: Session 7
Old vs. new beach alternative indicator results
E. coli cntcrococci BacHuman Lachno2 Gull2
Conclusions
Next generation sequencing reveals
Bacteroides and Lachnospiraceae are rich
in host indicators
I lighly abundant in gulls Catellicoccus
marimommalium the most abundant taxon
among all samples.
Sequencing water samples takes advantage
of complex profiles
qPCR for new targets can show
sources and quantify signal
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U.S. EPA's 2016 Recreational Waters Conference
Question & Answer Session
Question 1
Ali Boehm: Why are we measuring Gen-Bac [general bacteroides marker]? They are not part of the
criteria.
Answer 1
Sandra McLellan: Our lab stopped measuring Gen-Bac several years ago. We found it in sev-
eral samples along Lake Michigan. In the last 10 years we have seen other organisms that can
serve that same purpose. You still want some kind of qPCR for all bacteroides, though.
Answer 1 (follow-up)
Mia Mattioli: I agree. We didn't look for it. We did the decay rates though; they decay simi-
larly to the human markers.
Answer 1 (follow-up)
Jodi Harwood: We use Gen-Bac as a control.
Question 2
Jodi Harwood: I have questions for Helena [Solo-Gabriele], do you analyze effects of temperature on
indicator bacteria? Gulf waters are so different from East Coast waters.
Answer 2
Helena Solo-Gabriele: We put all the data into one number. There seems to be an optimal tem-
perature that the microbes grow in. I think there is a temperature effect.
Question 2 (follow-up)
Jodi Harwood: We see high levels around vegetation. I am also wondering about the birds because they
tend to have high levels of enterococcus compared to fecal coliforms. For Asli [Asian]. Your signal
persisted?
Answer 2 (follow-up)
Asli Asian: We saw it right after a precipitation event.
Comment 2
Jodi Harwood: We have issues in Florida with deer feces; we can't use it because we have a lot
of deer.
Answer 2 (follow-up)
Sandra McLellan: That's one thing that prompted us to look for another marker. HF183 is
good, but we wanted a different one.
Question 3
(Unknown): Do you think the higher concentration in the sediment during the warmer season is
related to decay?
Answer 3
Asli Asian: It looks like it is related, but we need more research.
Question 4
Richard Whitman: For Helena [Solo-Gabriele]. The data seems to lend itself to regression tree/decision
tree. Did you try that?
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Day Two: Session 7
Answer 4
Helena Solo-Gabriele: We first broke the beaches into six categories. We didn't do it method-
ologically, though.
Question 5
Richard Whitman: Your results have tremendous implication for site selection of beaches and site
design. I have tried to get the Army Corps of Engineers to think about that and develop strategies for
when and where to remediate.
Answer 5
Helena Solo-Gabriele: I have looked a lot at bays versus open beaches. There is a value to bay
beaches, lots of characteristics that people like. But if you have it, you have to manage it to
minimize other sources of contamination, like not allowing dogs. You don't have as much
flushing there.
Question 6
Marirosa Molina: For Sandra [McLellan]. Can you comment on the aging of sewage? You have stand-
ing water in the pipe and other factors that could change things in the micro community.
Answer 6
Sandra McLellan: We have tried to look at that in several ways. That is why we wanted to
develop electrophoresis markers: they are gram positive and might be hardier in the envi-
ronment. Its acts a little like a stoichiometric clock. They disappear slightly differently. We
are hoping to do some plume studies next year and sample day after day to help us answer
those questions.
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